Department of Microbial, Biochemical
and Food Biotechnology
BOC 314
Molecular Biology
BIOCHEMISTRY (BOC 314): Molecular Biology
A. Lecturers:
Prof. J. Albertyn
Dr. B. Visser
B. Duration of course:
Duration of course:
Section A – Prof Albertyn, 23 January – 30 March
Section B – Dr. B. Visser, 10 April – 11 May
C. Lecture periods: Maandag (AFR)
Woensdag (AFR)
Wednesday (ENG)
Friday (ENG)
D. Practicals:
Office: MKBOC 51
Office: Biology Building 134
Period 2
Period 5
Period 6
Period 5
(08h10 – 09h00)
(11h10 – 12h00)
(12h10 – 13h00)
(11h10 – 12h00)
CRS 1
FGG 361
LCT F
BIB OUD (SASOL Library
Auditorium)
Computer lab E, 2-5 pm.
Attendance of all practical’s and submission of all reports and
all assignments are compulsory. Neglect thereof may lead to
refusal of exam admittance according to Regulation A14(c) of
the University of the Free State.
E. Prescribed book: Gene cloning & DNA analysis, 6th Edition, T.A. Brown
F. Tests:
Unannounced class tests / Computer aided tests
Semester test 1: 12 March 2011, 2-5pm (In practical time slot)
Semester test 2: 3 May 2011
Supplementary semester test: 8 May
THE VENUE AND TIME WILL BE CONFIRMED AT A LATER STAGE.
Mark Calculation:
Main/class tests:
Practical:
60%
40%
IT WILL BE ASSUMED THAT ANY ANNOUNCEMENTS MADE IN THE CLASS
HAVE BEEN HEARD BY EVERYONE. IT IS YOUR OWN RESPONSIBILITY TO
FIND OUT IF ANY ADDITIONAL NOTES OR INFORMATION HAVE BEEN
GIVEN IN CLASS!
ANNOUNCEMENTS WILL BE PLACED ON BLACKBOARD AND/OR VIA EMAIL. IT IS THUS IMPERATIVE TO ACCESS BOTH BLACKBOARD AS WELL
AS YOUR UNIVERSITY E-MAIL ACCOUNT FREQUENTLY.
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BOC314 (16 credits) - Molecular biology
Module content:
The emphasis of this course is placed on the cloning of genes from single-and multi-cellular
organisms using a variety of different molecular cloning techniques. Expression vectors, molecular
manipulation of genes and database mining will also be studied. The characterization of gene
expression in transgenic organisms will also be discussed.
Module outcomes:
After successful completion of the course, the student should;
a)
have a thorough knowledge of the modern methods used to isolate genetic material
from different sources,
b)
acquired the theoretical knowledge and practical skills to clone genes from both
single and multi-cellular organisms and be familiar with expression systems that are
used in recombinant DNA technology,
c)
be able to explain how the gene and the encoded protein can be characterised in
transgenic organisms and
d)
understand genomics and proteomics information-based biology
Assessment information
The semester mark for BOC314 will be calculated as follows:
 Section A – Prof. J. Albertyn
o This section counts a total of 66% of the overall course, this will be calculated
as follows:
 Multiple choice tests – 10%
 Semester test – 50%
 Practical assignments – 40%
 100% x 0.66 = 66%
 Section B – Dr. B. Visser
o This section counts a total of 33% of the overall course, this will be calculated
as follows:
 Semester test – 60%
 Practical assignments – 40%
 100% x 0.33 = 33%
 Semester tests
 Three semester tests are scheduled; no additional ‘sick’ test will be scheduled. You
need to write 2 of the 3 tests. However, take in account that the supplementary test
(scheduled for 8 May will include all the work covered in the course).
o
o
o
Semester test 1 – This test is scheduled for 12 March 2012 and will cover all the
work done in lecture 1-12.
Semester test 2 - This test is scheduled for 3 May 2012 and will cover all the
work done in lecture 13-24.
A supplementary test is scheduled for 8 May 2012. This test will cover all the
work done in the course (thus lecture 1-26).
Assessment command words:
The following are a set of command words that can appear during assessment. Ensure that you
understand what is expected from you when these words are used in a question.

Name/write down – only facts are required, short and to the point.

Describe – Here, performance is expected on a knowledge level. Properties, facts or
results should be provided in a logical, well-structured manner. No comment or reasoning
is required. Tell a story. A description suggests that you convey a mental image or give an
account of something.

Define - Reproduction of knowledge is required. The answer is a clear, to the point
(concise) description of a concept so that its meaning is clearly explained.

Explain - The case is presented in a straight forward manner so that the reader will clearly
understand the meaning of the explanation. This may require a definition but will also
require some development of the point or points being asked. Write a detailed answer that
covers how and why a thing happens.

Give an overview/outline - A large volume of knowledge needs to be systematically
summarized and conveyed in a logical without the essence of the issue being lost. Give
only the key facts of the topic. You may need to set out the steps of a procedure or process
– make sure you write down the steps in the correct order.

Using examples…/explain what is meant by... A definition of a key term required plus an
example - drawn from any evidence or the case study - helps to support the explanation.
Know your definitions!

Compare –Point out the similarities and the differences between two or more things.

Evaluate - You will be given some facts, data, or other kind of information. Write about the
data or facts and provide your own conclusion or opinion on them. Weigh arguments for
and against something; assess all evidence; decide which opinions, theories, models or
items are preferable.

Calculate - Work out a number. You may need to use an equation.

Discuss - Write about the issues related to a topic. You would be expected to put both
sides of a case or an issue/argument in your answer and to make some evaluative
comment about the factors you are discussing.

Always read exam questions carefully, even if you recognise the words used. Look at the
information in the question and the number of points that the question counts to see how
much detail the examiner is looking for. In many cases you can use bullet points or a
diagram if it helps your answer.
Remember the importance of proper language skills in your answer (e.g. ‘SMS language’
is not acceptable). Spelling, punctuation and grammar are important and, ensure that the
examiner can read your writing!

Module content, Learning objectives and assessment criteria
Section A – Prof. J. Albertyn
Gene cloning and analysis: 6th edition, T.A. Brown.
Lecture
number
Lecture content
Background lecture – what are DNA and RNA, basic steps of transcription and
translation.
1.
Chapter 1: Why Gene Cloning and DNA Analysis are Important
1.1 The early development of genetics
1.2 The advent of gene cloning and the polymerase chain reaction
1.3 What is gene cloning?
1.4 What is PCR?
1.5 Why gene cloning and PCR are so important
1.5.1 Obtaining a pure sample of a gene by cloning
1.5.2 PCR can also be used to purify a gene
1.6 How to find your way through this book
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Chapter 1: Learning objective:
 Have an appreciation for the historical roots of gene cloning
 Understand the basic procedures involved in gene cloning
2.
Chapter 1: Assessment criteria

You should be able to define and explain, in short or at length and with the aid of diagrams, the
significance of the following terms:
o Gene cloning, polymerase chain reaction, polymerase, plasmids.

As well as:
o Be able to describe and/or draw in detail the steps involved in gene cloning.
o Explain the process of PCR.
o Explain why PCR is so important in the process of gene cloning.
Chapter 2: Vectors for Gene Cloning: Plasmids and Bacteriophages
2.1 Plasmids
2.1.1 Size and copy number
2.1.2 Conjugation and compatibility
2.1.3 Plasmid classification
2.1.4 Plasmids in organisms other than bacteria
2.2 Bacteriophages
2.2.1 The phage infection cycle
2.2.2 Lysogenic phages
Gene organization in the  DNA molecule
The linear and circular forms of  DNA
M13—a filamentous phage
2.2.3 Viruses as cloning vectors for other organisms
3.
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Chapter 2: Learning objective:

Understand and know the structure, properties and use of plasmids and bacteriophages
Chapter 2: Assessment criteria

You should be able to define and explain, in short or at length and with the aid of diagrams, the
significance of the following terms:
o Plasmids, origin of replication, plasmid size, copy number, conjugation, compatibility,
bacteriophages, lysogenic phages.

As well as:
o Be able to explain what a plasmid is as well as the role of plasmid size and copy number
in cloning procedures.
o Classify the different types of naturally occurring plasmids.
o Describe the phage infection cycles.
o Know and be able to describe the properties and use of phage  as well as phage M13.
5.
6.
Chapter 3: Purification of DNA from Living Cells
3.1 Preparation of total cell DNA
3.1.1 Growing and harvesting a bacterial culture
3.1.2 Preparation of a cell extract
3.1.3 Purification of DNA from a cell extract
Removing contaminants by organic extraction and enzyme digestion
Using ion-exchange chromatography to purify DNA from a cell extract
3.1.4 Concentration of DNA samples
3.1.5 Measurement of DNA concentration
3.1.6 Other methods for the preparation of total cell DNA
o
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3.2 Preparation of plasmid DNA
3.2.1 Separation on the basis of size
3.2.2 Separation on the basis of conformation
Alkaline denaturation
Ethidium bromide–caesium chloride density gradient centrifugation
3.2.3 Plasmid amplification
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3.3 Preparation of bacteriophage DNA
3.3.1 Growth of cultures to obtain a high  titer
3.3.2 Preparation of non-lysogenic  phages
3.3.3 Collection of phages from an infected culture
3.3.4 Purification of DNA from  phage particles
3.3.5 Purification of M13 DNA causes few problems
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Chapter 3: Learning objective:
 Understand and know the different methods to purify genomic and/or plasmid DNA, from a cell.
Chapter 3: Assessment criteria

You should be able to define and explain, in short or at length and with the aid of diagrams, the
significance of the following terms:
o DNA, bacterial culture, cell extract, defined medium, undefined medium, optical density,
ion-exchange chromatography, DNA concentration, measurement of DNA concentration,
plasmid conformation, plasmid amplification.
As well as:

o Be able to explain the stages involved in genomic DNA purification.
o Be able to discuss the different procedures used to purify either genomic or
plasmid DNA.
7.
8.
9.
Chapter 4: Manipulation of Purified DNA
4.1 The range of DNA manipulative enzymes
4.1.1 Nucleases
4.1.2 Ligases
4.1.3 Polymerases
4.1.4 DNA modifying enzymes
4.2 Enzymes for cutting DNA—restriction endonucleases
4.2.1 The discovery and function of restriction endonucleases
4.2.2 Type II restriction endonucleases cut DNA at specific nucleotide sequences
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4.2.3 Blunt ends and sticky ends
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4.2.4 The frequency of recognition sequences in a DNA molecule
4.2.5 Performing a restriction digest in the laboratory
4.2.6 Analysing the result of restriction endonuclease cleavage
Separation of molecules by gel electrophoresis
Visualizing DNA molecules in an agarose gel
4.2.7 Estimation of the sizes of DNA molecules
4.2.8 Mapping the positions of different restriction sites in a DNA molecule
4.2.9 Special gel electrophoresis methods for separating larger molecules
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4.3 Ligation—joining DNA molecules together
4.3.1 The mode of action of DNA ligase
4.3.2 Sticky ends increase the efficiency of ligation
4.3.3 Putting sticky ends onto a blunt-ended molecule
Linkers
Adaptors
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Producing sticky ends by homopolymer tailing
4.3.4 Blunt end ligation with a DNA topoisomerase
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Chapter 4: Learning objective:
 Understand how DNA can be manipulated.
Chapter 4: Assessment criteria

You should be able to define and explain, in short or at length and with the aid of diagrams, the
significance of the following terms:
o Recombination, DNA manipulative enzymes, blunts and sticky ends, gel electrophoresis,
restriction digest, ligation.

As well as:
o Be able to discuss in detail all aspect of manipulation of DNA using nucleases,
ligases, polymerases and/or other modifying enzymes.
o Be able to explain the purpose and use of restriction enzymes.
o Explain how one can determine the size of a DNA molecule.
Chapter 5: Introduction of DNA into Living Cells
5.1 Transformation—the uptake of DNA by bacterial cells
5.1.1 Not all species of bacteria are equally efficient at DNA uptake
5.1.2 Preparation of competent E. coli cells
5.1.3 Selection for transformed cells
5.2 Identification of recombinants
5.2.1 Recombinant selection with pBR322—insertional inactivation of an
antibiotic resistance gene
5.2.2 Insertional inactivation does not always involve antibiotic resistance
10.
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5.3 Introduction of phage DNA into bacterial cells
5.3.1 Transfection
5.3.2 In vitro packaging of  cloning vectors
5.3.3 Phage infection is visualized as plaques on an agar medium
5.4 Identification of recombinant phages
5.4.1 Insertional inactivation of a lacZ′ gene carried by the phage vector
5.4.2 Insertional inactivation of the  cI gene
5.4.3 Selection using the Spi phenotype
5.4.4 Selection on the basis of  genome size
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5.5 Introduction of DNA into non-bacterial cells
5.5.1 Transformation of individual cells
5.5.2 Transformation of whole organisms
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Chapter 5: Learning objective:
 Understand how DNA can be transformed into a living cell.
Chapter 5: Assessment criteria

You should be able to define and explain, in short or at length and with the aid of diagrams, the
significance of the following terms:
o Transformation, selection for transformed cells, identification of recombinants, insertional
inactivation.

As well as:
o List the different recombinant molecules that can be found in a ligation mixture.
o Be able to describe in detail the procedure(s) involved in transformation of DNA.
o Be able to describe in detail different ways to select and identify recombinants as
well as transformed cells.
o Describe the concept of insertional inactivation.
11.
Chapter 6: Cloning Vectors for E. coli
6.1 Cloning vectors based on E. coli plasmids
6.1.1 The nomenclature of plasmid cloning vectors
6.1.2 The useful properties of pBR322
6.1.3 The pedigree of pBR322
6.1.4 More sophisticated E. coli plasmid cloning vectors
pUC8—a Lac selection plasmid
pGEM3Z—in vitro transcription of cloned DNA
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6.2 Cloning vectors based on M13 bacteriophage
6.2.1 How to construct a phage cloning vector
6.2.2 Hybrid plasmid–M13 vectors
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6.5 Vectors for other bacteria
104
6.3 Cloning vectors based on  bacteriophage
6.3.1 Segments of the  genome can be deleted without impairing viability
6.3.2 Natural selection can be used to isolate modified 2 that lack certain restriction sites
6.3.3 Insertion and replacement vectors
Insertion vectors
Replacement vectors
6.3.4 Cloning experiments with  insertion or replacement vectors
6.3.5 Long DNA fragments can be cloned using a cosmid
6.4  and other high-capacity vectors enable genomic libraries to be constructed
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Chapter 6: Learning objective:
 Understand what a cloning vector is and how it can be used in E. coli.
Chapter 6: Assessment criteria

You should be able to define and explain, in short or at length and with the aid of diagrams, the
significance of the following terms:
o Cloning vector, pBR322, pUC8, pGEM-3Z, Blue-white selection.

As well as:
o Describe and discuss the properties and use of cloning vectors for E. coli.
12.
Review of Chapter 1-6
Chapter 7: Cloning Vectors for Eukaryotes
7.1 Vectors for yeast and other fungi
7.1.1 Selectable markers for the 2 µm plasmid
7.1.2 Vectors based on the 2 µm plasmid—yeast episomal plasmids
7.1.3 A YEp may insert into yeast chromosomal DNA
7.1.4 Other types of yeast cloning vector
7.1.5 Artificial chromosomes can be used to clone long pieces of DNA in yeast
The structure and use of a YAC vector
Applications for YAC vectors
7.1.6 Vectors for other yeasts and fungi
13.
7.2 Cloning vectors for higher plants
7.2.1 Agrobacterium tumefaciens—nature’s smallest genetic engineer
Using the Ti plasmid to introduce new genes into a plant cell
Production of transformed plants with the Ti plasmid
The Ri plasmid
Limitations of cloning with Agrobacterium plasmids
7.2.2 Cloning genes in plants by direct gene transfer
Direct gene transfer into the nucleus
Transfer of genes into the chloroplast genome
7.2.3 Attempts to use plant viruses as cloning vectors
7.3 Cloning vectors for animals
7.3.1 Cloning vectors for insects
P elements as cloning vectors for Drosophila
Cloning vectors based on insect viruses
7.3.2 Cloning in mammals
Viruses as cloning vectors for mammals
Gene cloning without a vector
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Chapter 7: Learning objective:
 Understand what a yeast cloning vector is and how it can be used.
Chapter 7: Assessment criteria

You should be able to define and explain, in short or at length and with the aid of diagrams, the
significance of the following terms:
o Cloning vector, 2µm plasmid, shuttle vectors, yeast episomal plasmid, yeast integrative
plasmid, yeast replicative plasmid, selectable marker, auxotrophic markers, homologous
recombination, transformation efficiency, yeast artificial chromosomes.

As well as:
o Describe the properties and applications of yeast cloning vectors.
o Explain how YAC’s can be used to clone long pieces of DNA.
14.
15.
Chapter 8: How to Obtain a Clone of a Specific Gene
8.1 The problem of selection
8.1.1 There are two basic strategies for obtaining the clone you want
8.2 Direct selection
8.2.1 Marker rescue extends the scope of direct selection
8.2.2 The scope and limitations of marker rescue
8.3 Identification of a clone from a gene library
8.3.1 Gene libraries
8.3.2 Not all genes are expressed at the same time
8.3.3 mRNA can be cloned as complementary DNA
8.4 Methods for clone identification
8.4.1 Complementary nucleic acid strands hybridize to each other
8.4.2 Colony and plaque hybridization probing
Labelling with a radioactive marker
Non-radioactive labelling
8.4.3 Examples of the practical use of hybridization probing
Abundance probing to analyse a cDNA library
Oligonucleotide probes for genes whose translation products have been
characterized
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Heterologous probing allows related genes to be identified
Southern hybridization enables a specific restriction fragment containing a
gene to be identified
8.4.4 Identification methods based on detection of the translation product of the
cloned gene
Antibodies are required for immunological detection methods
Using a purified antibody to detect protein in recombinant colonies
The problem of gene expression
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Chapter 8: Learning objective:
 Understand the experimental procedures involved in selection of a recombinant clone.
16.
17.
Chapter 8: Assessment criteria

You should be able to define and explain, in short or at length and with the aid of diagrams, the
significance of the following terms:
o Selection of a recombinant clone, marker rescue, gene library, clone identification,
labelling techniques, hybridization, primary and secondary antibody.

As well as:
o Be able to describe the different strategies that can be used to identify and/or
select a recombinant clone.
o Explain the different hybridization techniques that can be used to identify and/or
select a recombinant clone.
Chapter 9: The Polymerase Chain Reaction
9.1 The polymerase chain reaction in outline
9.2 PCR in more detail
9.2.1 Designing the oligonucleotide primers for a PCR
9.2.2 Working out the correct temperatures to use
9.3 After the PCR: studying PCR products
9.3.1 Gel electrophoresis of PCR products
9.3.2 Cloning PCR products
9.3.3 Problems with the error rate of Taq polymerase
9.4 Real-time PCR enables the amount of starting material to be quantified
9.4.1 Carrying out a quantitative PCR experiment
9.4.2 Real-time PCR can also quantify RNA
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Chapter 9: Learning objective:
 Have in-depth knowledge of the polymerase chain reaction.
Chapter 9: Assessment criteria

You should be able to define and explain, in short or at length and with the aid of diagrams, the
significance of the following terms:
o PCR, oligonucleotide primers, PCR products, Taq polymerase, error rate, Real-Time
PCR, quantitative PCR.

As well as:
o Be able to describe the use of the polymerase chain reaction in recombinant
DNA applications.
18.
Review of Chapter 7-9
Section B – Dr. B. Visser
During the final part of the course, the emphasis will fall on additional techniques whereby organisms can be
studied on DNA, RNA and protein levels. Students are advised to use the text book in combination with the
detailed notes that must be taken down in class. However, also make use of the Internet to get any
additional information that would help you to understand the work even better. I would therefore like to invite
you to attend every theory lecture, and come and see me with any problems that you might encounter.
1.2 Topics
Nr
Topic
Page number
1
2
3
4
5
6
7
DNA sequencing (normal and PCR based)
Genomics: Genome analysis and knockouts
Genomics: Antisense technology
Transcriptomics: Northern Blot
Transcriptomics: RT-PCR and Microarrays
Proteomics: Western blot / LC-MS analysis
Proteomics: Yeast two hybrid system
Chapter 10 – p 165
Chapter 12 – p 207
Chapter 12
Chapter 11 – p 185
Chapter 12 – p 215
Chapter 12 – p 217
Chapter 12 – p 220
Lecture
number
Lecture content
Part II The Applications of Gene Cloning and DNA Analysis in Research
10 Sequencing Genes and Genomes
10.1 The methodology for DNA sequencing
10.1.1 Chain termination DNA sequencing
Chain termination sequencing in outline
Not all DNA polymerases can be used for sequencing
Chain termination sequencing requires a single-stranded DNA
template
The primer determines the region of the template DNA that will be
sequenced
10.1.2 Pyrosequencing
Pyrosequencing involves detection of pulses of chemiluminescence
Massively parallel pyrosequencing
10.2 How to sequence a genome
10.2.1 The shotgun approach to genome sequencing
The Haemophilus influenzae genome sequencing project
Problems with shotgun sequencing
10.2.2 The clone contig approach
Clone contig assembly by chromosome walking
Rapid methods for clone contig assembly
Clone contig assembly by sequence tagged site content analysis
10.2.3 Using a map to aid sequence assembly
Genetic maps
Physical maps
The importance of a map in sequence assembly
12 Studying Genomes
12.1 Genome annotation
12.1.1 Identifying the genes in a genome sequence
Searching for open reading frames
Simple ORF scans are less effective at locating genes in eukaryotic
genomes
Gene location is aided by homology searching
Comparing the sequences of related genomes
12.1.2 Determining the function of an unknown gene
Assigning gene function by experimental analysis requires a reverse
approach to genetics
Specific genes can be inactivated by homologous recombination
12.2 Studies of the transcriptome and proteome
12.2.1 Studying the transcriptome
Studying a transcriptome by sequence analysis
Studying transcriptomes by microarray or chip analysis
12.2.2 Studying the proteome
Separating the proteins in a proteome
Identifying the individual proteins after separation
12.2.3 Studying protein–protein interactions
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Phage display
The yeast two hybrid system
11 Studying Gene Expression and Function
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11.1 Studying the RNA transcript of a gene
11.1.1 Detecting the presence of a transcript and determining its nucleotide
sequence
11.1.2 Transcript mapping by hybridization between gene and RNA
11.1.3 Transcript analysis by primer extension
11.1.4 Transcript analysis by PCR
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11.2 Studying the regulation of gene expression
11.2.1 Identifying protein binding sites on a DNA molecule
Gel retardation of DNA–protein complexes
Footprinting with DNase I
Modification interference assays
11.2.2 Identifying control sequences by deletion analysis
Reporter genes
Carrying out a deletion analysis
11.3 Identifying and studying the translation product of a cloned gene
11.3.1 HRT and HART can identify the translation product of a cloned gene
11.3.2 Analysis of proteins by in vitro mutagenesis
Different types of in vitro mutagenesis techniques
Using an oligonucleotide to create a point mutation in a cloned gene
Other methods of creating a point mutation in a cloned gene
The potential of in vitro mutagenesis
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Outcomes: Practical’s
Practical 1:
 Learn to retrieve specific sequences and identify given parameters (e.g. -10 &
-35 regions, termination region, translation start & stop codons etc.)
 Make predications regarding the strength of promoter based on consensus sequences as
given for known transcription factors
 Retrieve scientific articles regarding the specific gene, read through whole article (or only
abstract) and draw conclusions regarding the function of the gene, circumstances under which
gene is expressed, etc.
 Be able to identify relevant article info including name, volume, authors, date and title of journal
 Website: http://www.ncbi.nlm.nih.gov
(ENTREZ)
Practical 2:
 Use the specific name of a known gene and search for other genes showing high homology to
the known using the BLAST (Basic Local Alignment Search Tool) program
 Website: http://www.ncbi.nlm.nih.gov
(BLAST)
 Once homologous genes have been identified, the respective nucleotide & amino acid
sequences can be retrieved. This data (each specific homolog) also includes information and
parameters as was retrieved in Practical 1
 Once a number of nucleotide & amino acid sequences have been retrieved, they can be
aligned (either all the nucleotide sequences, or all the amino acid sequences, with each other)
using the ClustalW software
 The ClustalW alignment compares e.g. the amino acid sequences of 4 different proteins, and
highlights the similarities or differences between each of the amino acids in each of the
sequences
 Website: http://www2.ebi.ac.uk
(ClustalW)
 From these comparisons (alignment) one can draw conclusions regarding the function and/or
structure of an unknown gene by comparing it with the known gene and/or protein (of which
the function and structure have been published on a database) – As discussed in Part A1
 Also keep in mind that to understand the results obtained from a database, one should first
fully understand what the various symbols, parameters etc. mean (these are always give on
the particular website)
Practical 3:
 Here again the nucleotide sequence of a particular known gene is retrieved (ENTREZ)
 A program is used to translate the DNA nucleotide sequence into the amino acid sequence
(Universal genetic code)
 Website: http://tw.expasy.org
(Translate DNA -> Protein)
 Remember there are introns in Eukaryotic genes, so we have to remove them
 Use ENTREZ results to identify exons (e.g.):
mRNA
join(<205..452, 519..818 etc etc)
This means nucleotides 205 to 452 is an exon, and nucleotides 453 to 518 is an intron
 The spliced, mature mRNA nucleotide sequence can then again be translated into amino acid
sequence
 The derived amino acid sequence (after splicing) can then again be compared with the amino
acid sequence given in the ENTREZ results – again use ClustalW for the alignment
Practical 4:
 Once the spliced nucleotide sequence of a gene is know, primers can be designed
 Website: http://signal.salk.edu
(iSect Tools)



These primers can be used to BLAST (as in practical 2) for sequences which the primers may
“pick up”
If the primers appear to have a high specificity for the unknown gene, they can be used to PCR
up the gene
Remember that if the introns are not removed from the sequence, this could lead to one
designing primers within the intron region. And as the introns are usually removed by the
spliceosome, this would make no sense (thus we do the exercise of designing primers before
and after splicing so that the differences can be highlighted)
Practical 5:
 To confirm a PCR product, allow cloning of a fragment etc., the restriction pattern should be
known.
 Website: http://www.restrictionmapper.org/
 A restriction map indicates the position as well as the number of digest sites of any given
restriction enzyme.
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In summary:
Unknown gene

BLAST for homology

Align (ClustalW) homologous gene sequence to infer possible function for unknown by comparing
similarities and/or differences to a known source

Remove introns by interpreting ENTREZ results

Design PCR primers for amplification of the gene

(Translate spliced gene sequence into amino acid sequence and compare the unknown with the
known. Infer function and/or structure)

Determine the restriction profile of various restriction enzymes.